The main mechanism of intercellular communication between neurons is synaptic transmission. The central event in synaptic transmission is the release of neurotransmitter from the presynaptic terminal by Ca2+- triggered synaptic vesicle exocytosis. Despite extensive characterization of the proteins of synaptic vesicles, their role in the exocytotic event is poorly understood. The long term goal of the research proposed is to obtain a full understanding of the mechanism of exocytosis at the synapse by elucidating the three-dimensional structures of the proteins involved, and by characterizing the interactions between them. Among the proteins of synaptic vesicles, synaptotagmin is particularly interesting because: i) it binds phospholipids in a Ca2+-dependent manner; ii) it binds to several proteins of the presynaptic plasma membrane, including the neurexins; iii) several functional and genetic studies have shown that synaptotagmin is involved in Ca2+-evoked exocytosis. The specific aims of this proposal are directed to study the role of synaptotagmin in exocytosis by analyzing the three dimensional structure of its cytoplasmic region, and its interactions with Ca2+, with phospholipids and with neurexin 1alpha. NMR spectroscopy will be used to study the structure of the cytoplasmic region (39.3 kDa). First, the structure and Ca2+-induced conformational changes of each of the two central domains (C2 domains, Ca. 14 kDa each), which presumably are responsible for Ca2+-dependent binding to phospholipids, will be determined. Building on the structures obtained for the C2 domains, the conformation of larger fragments (18-22 kDa) will then be analyzed to elucidate the structure of the N-terminal and C-terminal domains, and to determine the relative orientations between all four domains. For this purpose, NMR experiments will also be performed on 15N- labeled samples of isolated domains in the presence of other unlabeled synaptotagmin fragments. Interactions with phospholipids will be studied by fluorescence and NMR spectroscopies using fluorescent- and spin-labeled lipids, as well as mixed lipid/detergent micelles. The binding mode of synaptotagmin to the cytoplasmic region of neurexin 1alpha (7 kDa) will be studied by determining its solution structure in the presence of different synaptotagmin fragments, also using combinations of 15N-labeled and unlabeled samples. Studies on different isoforms of synaptotagmin will be carried out to identify structural features responsible for functional differentiation. The success of the research proposed will represent an important methodological advance in structure determination of proteins in solution. The insights that this research will bring are relevant to the understanding of all neural functions, and to the development of therapies for diseases of the nervous system such as Parkinson's disease, myasthenia and schizophrenia.